The heart is a high-energy demand organ and ischemic heart disease is a significant cause of morbidity and mortality. During ischemia, macroautophagy (hereafter referred to as autophagy) is induced to sequester intracellular contents and organelles including mitochondria for lysosomal degradation resulting in preservation of cellular energy status and conferring cell survival. Mitochondrial autophagy (mitophagy) is a selective type of autophagy which also plays a protective role by eliminating compromised mitochondria, thereby limiting the activation of mitochondrial cell death pathways as well as supplying an energy source. Hexokinase-II (HK-II) catalyzes the first step of glycolysis, phosphorylating glucose, and is the predominant isoform in the heart. HK-II is increasingly recognized as a survival signaling nexus, especially as a protective molecule at mitochondria against acute oxidative stress such as reperfusion injury. Building on our recently published and preliminary data, this study will examine a new facet of HK-II survival signaling: the coordination of metabolic status with mitophagy and autophagy to enhance cell survival during metabolic suppression. Aim 1 will determine whether dissociation mitochondrial HK-II (mitoHK-II) plays a regulatory role in recognition of damaged mitochondria to limit cardiac damage induced by ischemia in cardiomyocytes using adenoviral expression of mitoHK-II dissociating peptide (15NG). We will determine whether HK-II binds to mitofusin-2, Parkin receptors at mitochondria to regulate mitophagy. In Aim 2, we will determine whether HK-II regulates non-selective autophagy during ischemia in cardiomyocytes. Based on our recent study, we hypothesize that HK-II binds to and inhibits mTOR complex 1 (TORC1) to increase protective autophagy under ischemia and that this binding occurs at lysosome, a TORC1 activation site, in response to energy depletion. In Aim 3, we will determine whether mitophagic and autophagic effects of HK- II emerged during ischemia play a protective role against ischemic stress in vivo heart using 15NG transgenic mice generated in the lab as well as in vivo expression of HK-II mutants using adeno-associated virus serotype 9 (AAV9) technique. The long term goal of this proposal is to unveil a previously unrecognized link between HK-II and protective autophagy as a potential target for therapeutic intervention in heart diseases.